PCR for direct detection of indigenous uncultured magnetic cocci in sediment and phylogenetic analysis of amplified 16S ribosomal DNA

Abstract

PCR primers specific to the 16S ribosomal DNA (rDNA) of magnetic cocci were designed and used to amplify DNA from magnetically isolated magnetic cocci. The PCR products were subcloned by ligation into plasmid vector pCRII, and five clones containing approximately 270-bp fragments of amplified DNA were sequenced. The specific primers were also used to detect magnetic coccus 16S rDNA in environmental samples. Magnetic coccus 16S rDNA was amplified from the water column above sediment kept in an anoxic environment in the laboratory, but little was amplified from a water column kept in an oxic environment. These results suggest that magnetic cocci in the water column in an anoxic environment had migrated there from the sediment as a response to the microoxic or anoxic conditions, rather than having been present previously in a nonmagnetic form and having become magnetic due to these conditions. The specific primers were also used to detect magnetic cocci in aquatic sediment. DNA was extracted from sediment by direct lysis and purified for use as a PCR template by electrophoresis on an agarose-polyvinylpyrrolidone gel. 16S rDNA was then amplified and subcloned, and two clones were sequenced. The clones were screened for chimeric DNA by comparing sections of each with the GenBank database.

Does capillary racetrack-based enrichment reflect the diversity of uncultivated magnetotactic cocci in environmental samples?

“…GM3F and GM4R). For comparison, the metagenome-based PCR approach (Thornhill et al, 1995;Zhou et al, 1996;Handelsman et al, 1998) was used to study the phylogenetic diversity of cocci in the same sample. Sequences belonging to magnetotactic cocci acquired from the metagenome-based approach of this study are distributed in three clusters (Fig.…”

“…The recent development of the 'metagenome' of the environmental sample (Handelsman et al, 1998), which enables the exploration of the phylogenetic and metabolic diversity of microorganisms without cultivation, provides a promising opportunity to investigate MTB from the environmental samples. Thornhill et al (1995) designed a set of PCR primers that are specific to the 16S rRNA genes of magnetotactic cocci and used them to detect cocci in metagenome extracted from aquatic sediment by direct lysis. Recently, another study has examined the diversity of MTB in different microcosms by denaturing gradient gel electrophoresis (DGGE) analysis with metagenomic DNA from a sediment sample (Flies et al, 2005).…”

Magnetic, geochemical and DNA properties of highly magnetic soils in England

“…and 95 per cent sequence identity to R. sphaeroides 16S rRNA; the reverse primer was speci®c for Magnetospirillum sp. Primers were also constructed that were speci®c for magnetococci from published sequences (Thornhill et al 1995).…”

Phylogenetic analysis of the bacterial communities in marine sediments

“…Because agarose gel electrophoresis effectively removes humic and other enzyme inhibitory compounds from indigenous soil bacterial DNA (32), we pursued this approach for marine sediments. We also wanted intact, high-molecular-weight chromosomal DNA because it has been reported that chimeric PCR product formation increases substantially with increased template fragmentation (25,26,54). High-molecular-weight DNA can be isolated by gel electrophoresis.…”